CN112293411A - Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius - Google Patents
Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius Download PDFInfo
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to a low-temperature preservation solution for echinococcus intermedius sperms and a preservation method, belonging to the technical field of low-temperature preservation of aquatic animal sperms, wherein the low-temperature preservation solution comprises 0.02g/L of florfenicol, 0.02g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride in percentage by mass. The low-temperature preservation solution and the method can improve the survival time of the sperms of the echinococcus intermedia, and the number ratio of the low-temperature preservation solution to the sperms to the ova is 10000: the fertilization rate of 95.67 percent can be still achieved under the sperm-egg ratio of 1, and technical support is provided for development and improvement of the germplasm resources of the strongylocentrotus intermedius and genetic breeding research.
Description
Technical Field
The invention belongs to the technical field of low-temperature preservation of aquatic animal sperms, and particularly relates to a low-temperature preservation liquid and a preservation method for echinococcus intermedius sperms.
Background
Strongylocentrotus intermedius belongs to echinodermata, Hemicentripes, Strongylocentrotus, and Strongylocentrotus, is an excellent variety introduced from Japan, is the only type of strongylocentrotus which can be used for full-artificial breeding, and is also the main type of strongylocentrotus intermedius culture in China.
The sperm preservation technology has important significance for the breeding, the improved variety cultivation, the resource protection and the biological research of aquatic animals. In the germplasm preservation and improved breed breeding work of sea urchins, as new breeds need to be introduced or bred from different regions, the breeding periods of the sea urchins in different regions are different, the death of the sea urchins is easy to occur due to long-distance transportation, and the gonads of the male and female parent sea urchins are asynchronous in development, the semen needs to be preserved in scientific research and production practice. The sperm preservation technology is generally low-temperature preservation and ultralow-temperature freezing preservation, and the low-temperature preservation principle is that the low temperature can reduce the metabolic activity of the sperm, so that the service life of the sperm is prolonged for several days or more than ten days; the ultra-low temperature cryopreservation is to make the sperm at-196 ℃, the metabolism and movement of the sperm completely stop, and the life can be preserved for a long time in a static state. Compared with cryopreservation, the low-temperature (4 ℃) storage of the sperms has the advantages of simple operation, easy satisfaction of storage conditions and strong repeatability, and can also prevent the ultrastructure damage caused by the formation of ice crystals in the cells due to quick freezing. At present, the researches on the cryopreservation of the sperm of the aquatic economic fishes are more in China, and the cryopreservation technology of the sperm of echinoderm, especially the echinoderm, has not been reported yet.
Disclosure of Invention
The invention aims to provide a low-temperature preservation solution and a preservation method for echinus intermedia sperms, which can improve the survival time of the echinus intermedia sperms and provide technical support for developing and improving echinus germplasm resources and researching genetic breeding.
In order to solve the technical problems, the invention adopts the following technical scheme:
the low-temperature preservation solution for the sperms of the echinococcus intermedius comprises, by mass, 0.02g/L of florfenicol, 0.02g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride;
the sperm activator comprises 0.7% of NaCl, 0.05% of KCl and 0.2% of CaCl in percentage by mass20.02% NaOH and 1.5% glucose.
The invention also provides a method for preserving the sperms of the Strongylocentrotus intermedius by using the low-temperature preservation solution, which comprises the following steps:
1) collecting the semen of the sea urchin with the middle ball in a centrifuge tube, centrifuging for five minutes at 4 ℃ at 1000r/min, and removing the supernatant;
2) diluting centrifuged Hemicentrotus Seu Strongylocentrotus seminal plasma with 20 μ g/ml low temperature preservation solution to 100 times of original volume, centrifuging at 4 deg.C and 1000r/min for 5min, and removing supernatant; adding 20 mug/ml of low-temperature preservation solution with the same volume as the supernatant, and centrifuging again; repeating the operation for 2-3 times;
3) after the last centrifugation operation is finished, adding 20 mug/ml of low-temperature preservation solution with the same volume as the supernatant, marking the date on a centrifugal tube, and then putting the centrifugal tube into a refrigerator at 4 ℃ for sperm preservation;
4) when the sperm is used after preservation, the low-temperature preservation solution and the sperms are mixed and shaken evenly, and the sperm activating agent with the same volume is added to activate the sperms and combine the activated sperms with fresh ova to finish fertilization.
Further, the step 1) is to lightly wipe the surface of the sea urchin after injecting 0.5mol/L KCL solution near the peristomal membrane of the sea urchin intermedia to stimulate the sea urchin intermedia to discharge seminal fluid.
Furthermore, when the sperms of the echinococcus intermedius are collected, the seminal plasma collected in the first step is stored at the temperature of 14 ℃ due to different spermatogenesis time, so that the reduction of the vitality of the seminal plasma is avoided.
Further, the sperm activating agent comprises 0.7 percent of NaCl, 0.05 percent of KCl and 0.2 percent of CaCl in percentage by mass20.02% NaOH and 1.5% glucose.
Further, the echinus intermedia sperm preserved by the low-temperature preservation solution is used within 30 days.
Further, step 3) should avoid shaking in the sperm preservation process, the centrifuging tube that keeps the sperm need keep sealed.
Further, the ratio of the number of the low-temperature preservation solution to the number of the sperms and the ova in the step 4) is 10000: the fertilization rate of 95.67 percent can be still achieved under the sperm-egg ratio of 1.
Detailed Description
The technical solution of the present invention is further explained by the following examples, but the scope of the present invention is not limited in any way by the examples.
Example 1
1. Screening of low-temperature preservation solution of echinococcus intermedius sperm
1.1. Preparing and preserving sperm preserving fluid:
preparing sterile seawater, filtering natural seawater with 0.2 μm microporous membrane, and storing in sterilized conical flask.
Preparing low-temperature preservation solution, weighing the low-temperature preservation solution reagent, placing the low-temperature preservation solution reagent in sterile seawater to prepare 20 mu g/ml low-temperature preservation solution, uniformly mixing the low-temperature preservation solution with a magnetic stirrer, filtering the mixture through a 0.2 mu m microporous filter membrane, and preserving the mixture in a low-temperature refrigerator at 4 ℃.
Low-temperature preservation solution a: filtering seawater as a control group;
low-temperature storage liquid B1: 0.04g/L of florfenicol, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature storage liquid B2: 0.02g/L of florfenicol, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature storage liquid B3: 0.01g/L of florfenicol, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature preservation solution C1: doxycycline 0.04g/L, sodium chloride 26.72g/L, potassium chloride 0.72g/L, calcium chloride 1.15g/L, sodium bicarbonate 0.20g/L, magnesium chloride 2.26g/L
Low-temperature preservation solution C2: doxycycline 0.02g/L, sodium chloride 26.72g/L, potassium chloride 0.72g/L, calcium chloride 1.15g/L, sodium bicarbonate 0.20g/L, magnesium chloride 2.26g/L
0.01g/L of low-temperature preservation solution C3 doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature storage liquid D1: 0.02g/L of florfenicol, 0.02g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature storage liquid D2: 0.01g/L of florfenicol, 0.01g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
Low-temperature storage liquid D3: 0.005g/L of florfenicol, 0.005g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride
The sperm activator comprises 0.7% of NaCl, 0.05% of KCl and 0.2% of CaCl in percentage by mass20.02% NaOH and 1.5% glucose.
1.2. Observation of sperm motility
A small amount of semen is uniformly mixed with natural seawater, and the state of the sperm is immediately observed under a microscope, and the sperm motility is evaluated by taking the swinging frequency, the movement speed, the movement direction and the like of the sperm as indexes. Sperm motility is positively correlated with insemination capability, and the higher the sperm motility, the stronger the insemination capability. The sperm cells were stained with 5g/L trypan blue solution at room temperature for 15min and observed for sperm cell death under the microscope's mirror. The apical or nucleosomal part of dead sperm is blue in color and live sperm is not stained. Each time, 200 sperm cells were counted and repeated 3 times. Calculating the sperm mortality rate:
sperm mortality (%) × (number of stained sperm/total sperm) × 100%.
Sperm survival (%)% 1-sperm mortality%
And mixing the activated sperms and fresh ova for fertilization, determining the sperm-egg ratio, counting 100 ova each time, repeating for 3 times, and calculating the fertilization rate. The fertilization rate was expressed as a percentage of the total statistics of the number of eggs that developed into gastrula. The survival rate of the fresh sperms selected by the test is over 90 percent.
3. Data analysis
And (3) analyzing the data of the experimental result by using SPSS 18.0 software, analyzing by using one-factor analysis of variance, and comparing afterwards if the difference has statistical significance, wherein the SNK method is used for the variance simultaneously, the Duncan's method is used for the variance simultaneously, and the difference P <0.05 has statistical significance.
The results show that the survival rate of sperm is greatly reduced at 7d when the preservative solution A is a seawater filtering control group, while the experimental group taking B, C, D as a protective solution can still enable most sperm to survive. After a shelf life of 30d, sperm survival for each group is shown in table 1: the D1 preservative fluid has the best effect, the sperms in the rest preservative fluids almost all die after B2 times.
TABLE 1100 dilution times survival rate of sperm of sea urchin under low-temperature storage condition of 4 deg.C
Marking different letters indicates that the difference is statistically significant (P <0.05), marking the same letters indicates that the difference is not statistically significant (P >0.05)
4. Verification of artificial insemination effect
After the sea urchin sperms are stored at the low temperature of 4 ℃ for 30 days, the sea urchin sperms in the D1 storage liquid are fully mixed with the storage liquid, the sperm activating agent with the same volume is added to activate the sperms, the sperms are combined with the mature eggs of the Strongylocentrotus intermedius, the combination of the fresh sea urchin sperms and the mature eggs of the Strongylocentrotus intermedius is used as a control, the fertilization rates under different sperm-egg ratios are recorded, and the experimental results are shown in table 2: the ratio of the number of sperms to the number of eggs is 10000: 1, the fertilization effect of fertilizing 95.67 percent of the ovum and developing to the blastocyst stage, namely, preserving the semen in sufficient quantity can reach the level of fresh sperms.
TABLE 2 fertilization rates of sea urchin sperms stored under different conditions
The preservation effect of the sperm cryopreservation mode and the preservation effect of different antibiotic preservation solutions is shown, and the D1 preservation condition has a better preservation effect. By low temperature storage for up to 30 days, at 10000: the fertilization rate of 95.67 percent can be still achieved under the sperm-egg ratio of 1.
Example 2
(1) Preparing sterile seawater: collecting natural seawater filtered by precipitation, filtering with 0.2 μm microporous membrane, sterilizing with high temperature steam sterilizing pot, storing in 1000ml conical flask, and sealing with tinfoil paper.
(2) Preparing low-temperature preservation solution of echinococcus intermedius sperms: accurately weighing the medicament by using an electronic balance, pouring the medicament into 1000ml of sterile seawater, wherein the components and the mass percentage of the low-temperature preservation solution are 0.02g/L of florfenicol, 0.02g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride. All the components are mixed with seawater uniformly by using a magnetic stirrer, filtered by a 0.2 mu m microporous filter membrane and stored in a low-temperature refrigerator at 4 ℃.
(3) Taking healthy intermediate sea urchin with good gonad development, injecting 1-2ml of 0.5ml/LKCL solution into the peristomal membrane, wiping the surface of the sea urchin with absorbent paper, and pouring the sea urchin into a culture dish. After the sea urchins produce sperms, collecting the sperms in a 5ml centrifuge tube by using a Pasteur pipette, and temporarily storing the sperms in ice cakes to keep the temperature low.
(4) After the semen is completely collected, placing the semen in a centrifuge, centrifuging the semen for five minutes at the temperature of 4 ℃ at the speed of 1000r/min, and removing the supernatant.
(5) Taking 10 μ L of centrifuged Hemicentrotus Seu Strongylocentrotus seminal plasma in 1mL centrifuge tube with 1000 μ L pipette, adding 990 μ L of sperm preservation solution, repeatedly blowing and beating, and mixing. Centrifuging at 4 deg.C and 1000r/min for five minutes, discarding supernatant, and repeating the operation for three times.
(6) And after the last centrifugation operation, adding the sperm low-temperature preservation solution with the volume same as that of the supernatant into a centrifuge tube to enable the volume to reach 1ml of the scale mark of the centrifuge tube, and preserving in a low-temperature refrigerator at 4 ℃ to finish the sperm low-temperature preservation operation of the echinus intermedia, wherein the vibration is avoided in the preservation process, and the centrifuge tube for preserving the sperm needs to be sealed.
(7) When the sperm preserving liquid is used, the sperm preserving liquid and the sperms are mixed and shaken evenly, the sperm activating agent with the same volume is added, the sperm movement activity is successfully activated after the observation under a microscope, and the activated sperm is combined with the fresh ovum as soon as possible to finish the fertilization.
And after preserving at 4 ℃ for 30 days, taking the sea urchin semen preserved by the preservation solution, activating by a sperm activating agent, adding the sea urchin semen into a fresh egg, and counting the fertilization rate of the sea urchin egg. The result shows that the preserved sea urchin sperms have fertilization capability, and the fertilization rate of the eggs can reach 95.67% by adding enough sperms, and the eggs can normally develop to the blastocyst stage.
Claims (8)
1. The low-temperature preservation liquid for the sperms of the echinococcus intermedius is characterized by comprising 0.02g/L of florfenicol, 0.02g/L of doxycycline, 26.72g/L of sodium chloride, 0.72g/L of potassium chloride, 1.15g/L of calcium chloride, 0.20g/L of sodium bicarbonate and 2.26g/L of magnesium chloride in percentage by mass.
2. The method for preserving sperm of Strongylocentrotus intermedius with the cryopreservation solution of claim 1, comprising the steps of:
1) collecting the semen of the sea urchin with the middle ball in a centrifuge tube, centrifuging for five minutes at 4 ℃ at 1000r/min, and removing the supernatant;
2) diluting centrifuged Hemicentrotus Seu Strongylocentrotus seminal plasma with 20 μ g/ml low temperature preservation solution to 100 times of original volume, centrifuging at 4 deg.C and 1000r/min for 5min, and removing supernatant; adding 20 mug/ml of low-temperature preservation solution with the same volume as the supernatant, and centrifuging again; repeating the operation for 2-3 times;
3) after the last centrifugation operation is finished, adding 20 mug/ml of low-temperature preservation solution with the same volume as the supernatant, marking the date on a centrifugal tube, and then putting the centrifugal tube into a refrigerator at 4 ℃ for sperm preservation;
4) when the sperm is used after preservation, the low-temperature preservation solution and the sperms are mixed and shaken evenly, and the sperm activating agent with the same volume is added to activate the sperms and combine the activated sperms with fresh ova to finish fertilization.
3. The method according to claim 2, wherein the step 1) is performed by gently rubbing the surface of the sea urchin after injecting 0.5mol/L KCL solution near the peristomal membrane of the sea urchin intermedia to stimulate the sea urchin intermedia to discharge seminal fluid.
4. The method according to claim 2, wherein the sperm of the Strongylocentrotus intermedius is collected, and the seminal plasma collected in the previous step is preserved at 14 ℃ for different spermatogenesis time, so as to avoid the reduction of the activity.
5. The method of claim 2, wherein the sperm activating agent comprises 0.7% NaCl, 0.05% KCl, 0.2% CaCl20.02% NaOH and 1.5% glucose.
6. The method according to claim 2, wherein the use of the cryopreservation solution preserved Hemicentrotus intermedia sperm is within 30 days of use.
7. The method of claim 2, wherein the sperm of step 3) is preserved without shaking, and the tube for storing the sperm is sealed.
8. The method according to claim 2, wherein the ratio of the number of sperm to egg of the cryo-preservation solution of step 4) is in the range of 10000: 1.
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| CN202011363847.8A CN112293411B (en) | 2020-11-27 | 2020-11-27 | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius |
| AU2021104255A AU2021104255A4 (en) | 2020-11-27 | 2021-07-16 | Hypothermic preservation solution and method for strongylocentrotus intermedius sperm |
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| CN113273567A (en) * | 2021-06-04 | 2021-08-20 | 大连海洋大学 | Low-temperature preservation liquid for patinopecten yessoensis sperms and preservation and use method |
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